RNA molecules with structure-dependent functions play important roles throughout molecular biology, and the broad, long-term objective of this grant is to understand these roles. The experiments of this proposal focus on the mechanism, biology, and evolution of RNA interference (RNAi), an example of a structure-dependent function in which double-stranded RNA (dsRNA) triggers the destruction of corresponding cellular mRNAs. Although RNAi has been lost in Saccharomyces cerevisiae, it is present in other budding yeasts, including Saccharomyces castellii (a close relative of S. cerevisiae) and Candida albicans (a common human pathogen). These species use noncanonical Dicer proteins to process long dsRNA into small interfering RNAs (siRNAs), which are loaded into the Argonaute protein to direct silencing. Introducing Dicer and Argonaute of S. castellii restores RNAi in S. cerevisiae, and the reconstituted pathway silences endogenous retrotransposons. The discovery of RNAi in budding yeast opens new opportunities for exploring the mechanism, biology, and evolution of the pathway. The first specific aim of the proposed experiments is to determine the consequences of losing or restoring RNAi. Methods will include phenotypic profiling, small-RNA sequencing, and mRNA sequencing. Endogenous dsRNA elements known as Killer elements also will be monitored, with the expectation that their retention will be compromised in the RNAi-reconstituted S. cerevisiae strain. These experiments will explore how cells cope with the introduction of a new gene-regulatory pathway and provide insight into why RNAi was lost in some species. The second aim is to identify additional components of RNAi in budding yeast. Methods will include genetic selections and mass spectrometry of co-immunoprecipitated proteins. Identifying new components and modifiers would be important not only for understanding the yeast pathway but could shed light on RNAi pathways in plants and animals, including humans. Results of both this aim and aim 1 will also be of practical interest for those using RNAi-reconstituted strains to study gene function in S. cerevisiae and those attempting to port RNAi into other RNAi-deficient organisms. The third aim is to determine the mechanism of RNAi in budding yeast. To test the hypothesis that silencing is post- transcriptional, RNAi-mutant strains will be monitored for changes in RNA Polymerase II binding, mRNA turnover, and the correspondence between sequenced siRNAs and mRNA degradation fragments. Biochemical and structural experiments will also test a proposed mechanism for how purified budding-yeast Dicer produces 23-nt siRNAs, despite lacking the protein domain that canonical Dicers require for this activity. Experiments of all three aims will leverage, for the first time, the powerful tools of budding yeast for the study of RNAi. A thorough understanding of this recently identified pathway in budding yeast should provide important insights regarding RNAi and related gene-silencing pathways in other eukaryotes and thereby contribute to fundamental knowledge relevant to human health.